C2 and PH domains are ubiquitous eukaryotic targeting motifs that drive membrane docking in response to a second messenger. When activated by second messenger signals, signaling proteins containing these domains dock to specific target membranes and control a wide array of essential cellular pathways. This continuing project investigates the complex signaling circuit at the leading edge of polarized cells, where C2 and PH domains play a dominant role by targeting dozens of signaling proteins to the appropriate membrane surface. The resulting targeting is universally required for cell migration and chemotaxis: the present focus is the leading edge of leukocytes that drive the primary immune response. The membrane targeting reaction of a given C2 or PH domain is optimized to ensure that the domain docks with exquisite specificity, as well as the appropriate affinity and speed, to its target membrane. Subsequently, the lipid-bound domain diffuses randomly in the bilayer plane for the total time and distance needed to undergo productive collisions with effector molecules. Finally, the domain dissociates to terminate activity. One broad goal of this continuing proposal is to elucidate the fundamental molecular mechanisms underlying the membrane targeting reactions of key leading edge C2 and PH domains. The Specific Aims begin with in vitro studies of isolated C2 and PH domains, then move to in vitro studies of full length proteins, and finally to studies of domains and proteins in live cells. Aim 1 employs spin label EPR measurements to elucidate the first membrane docking geometries of PH domains. Aim 2 analyzes the membrane targeting mechanisms of representative C2 and PH domains, and tests a new working model for the protein-lipid interactions that control targeting. Aim 3 employs innovative single molecule methods to dissect the contributions of multiple membrane docking domains in full length proteins, and to analyze the regulatory mechanisms of full length proteins. Aim 4 tests the conclusions of in vitro studies in live cells, and develops new biosensors to analyze second messenger signals at the leading edge of polarized cells. Completion of these Aims will reveal key principles of membrane recognition in cell migration, the immune response inflammation, and cancer. PUBLIC HEALTH RELEVANCE: This proposal investigates the ubiquitous C2 and PH domain motifs that regulate the membrane targeting of signaling proteins in a wide array of cellular pathways. Certain C2 and PH domains also play central roles in human diseases such as inflammation and cancer; for example, the highly oncogenic E17K mutation of AKT1 PH domain drives constitutive plasma membrane targeting by an unidentified mechanism, yielding kinase superactivation that accounts for its carcinogenicity. Preliminary results reveal that the E17K mutation enhances membrane targeting by changing the target lipid specificity of the PH domain, thereby defining the molecular mechanism of this important oncogenic mutation.